Protein glycosylation is a critical quality attribute in determining the potency and pharmacokinetic longevity of a biologic. Understanding how each glycosylation site affects the overall function of the protein helps optimize final product quality. By achieving a homogeneous glycosylation profile, increases in yield can be achieved with assurance of product comparability.
O-linked glycosylation refers to the attachment of one of the sugars N-acetylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine. O-linked sugar residues have fewer structural rules than N-linked glycosylation and therefore create a greater diversity in glycoforms. O-linked glycosylation is difficult to predict due to lack of consensus recognition sequences. However, neural network approaches have been developed to better predict mucin-type O-linked sites (Julenius, K., et.al. 2005. Glycobiology 15, 153-164). While most reports to date document the role of O-glycans in the glycoproteins' binding capability or the masking of its peptide backbone, one particular case study demonstrates that follicle-stimulating hormone (FSH) analogs with more O-linked sites are less bioactive than FSH analogs with increased N-linked glycosylation sites (Weenen, C. et al. 2004. J Clin Endocrinol Metab 89, 5204-5212). O-linked glycans have been shown to be non-essential for cell surface expression of glycoprotein gC-1 but may interfere with its epitope's binding domain and its binding capability (Biller, M. et al. 2000. Glycobiology 10, 1259-1269).
The presence of glycosylation is believed to affect immunogenicity, efficacy, solubility, and half-life of commercial biologics (Lis, H. et.al. 1993. Eur J Biochem 218, 1-27; Lowe, J. B. et.al. 2003. Annu Rev Biochem 72, 643-691; Van den Steen, P. et.al. 1998. Grit Rev Biochem Mol Biol 33, 151-208). However, most literature focuses on the effects of N-linked glycosylation (Hossler, P. et.al. 2009. Glycobiology 19, 936-949). The information regarding the effects of O-linked glycosylation on protein quality is scarce; especially with respect to protein aggregation.
Aggregation of a protein therapeutic during manufacture is undesirable and requires extensive downstream processing for its removal. Therefore, there is a need to minimize protein aggregation resulting in reduced manufacturing costs and accelerated process optimization. The discovery of the relationship between O-linked glycosylation and aggregation of a therapeutic glycoprotein enables the manufacturer to modify the therapeutic glycoprotein early in product development.